Nucleic acid circularization and amplification on a surface

1. A method of amplifying a single-stranded polynucleotide, the method comprising:

contacting a solid support with the single-stranded polynucleotide comprising a first primer binding sequence and a second primer binding sequence, and hybridizing said first primer binding sequence and said second primer binding sequence to a splint oligonucleotide attached to the solid support;

extending the first primer binding sequence and ligating the extended first primer binding sequence to the second primer binding sequence together to form a circular polynucleotide;

extending the splint oligonucleotide with a polymerase to generate an amplification product comprising a complement of the circular polynucleotide; and

hybridizing the amplification product to a second oligonucleotide attached to the solid support comprising a blocking group at a 3′ end that prevents polymerase extension.

2. The method of claim 1 , wherein said second oligonucleotide comprises the second primer binding sequence.

3. The method of claim 1 , wherein said single-stranded polynucleotide comprises a third primer binding sequence, and said second oligonucleotide comprises said third primer binding sequence.

4. The method of claim 1 , wherein said solid support is a bead or substantially planar.

5. The method of claim 1 , wherein said amplification product comprises a plurality of complements of the circular polynucleotide.

6. The method of claim 1 , wherein extending comprises incubating the splint oligonucleotide with a strand-displacing polymerase (a) for about 1 minute to about 2 hours, and/or (b) at a temperature of about 20° C. to about 50° C.

7. The method of claim 6 , wherein said strand-displacing polymerase is a phi29 polymerase, a phi29 mutant polymerase, or a thermostable phi29 mutant polymerase.

8. The method of claim 1 , wherein the circular polynucleotide is about 100 to about 1000 nucleotides or about 1000 to about 5000 nucleotides in length.

9. The method of claim 1 , wherein said amplification product comprises one or more cleavable sites.

10. The method of claim 1 , wherein said solid support comprises an array of wells.

11. The method of claim 1 , wherein said solid support comprises a plurality of splint oligonucleotides randomly distributed on the solid support.

12. The method of claim 1 , further comprising sequencing the amplification product.

13. The method of claim 1 , wherein said solid support comprises a polymer, wherein said splint oligonucleotide and second oligonucleotide are covalently attached to said polymer.

14. A method of forming a plurality of tethered amplification products on a solid support, said method comprising:

contacting a solid support with a sample comprising a plurality of single-stranded polynucleotides

hybridizing a first primer binding sequence and a second primer binding sequence of a single-stranded polynucleotide of said plurality of single-stranded polynucleotides to a splint oligonucleotide attached to the solid support, wherein said splint oligonucleotide comprises, from 5′ to 3′, a first primer sequence, a third sequence, and a second primer sequence, and;

extending the first primer binding sequence to form a complement of the third sequence and ligating the complement of the third sequence and the second primer binding sequence together to form a circular polynucleotide;

extending the splint oligonucleotide with a polymerase to generate an amplification product comprising a complement of the circular polynucleotide; and hybridizing the amplification product to a second oligonucleotide attached to the solid support, wherein said second oligonucleotide is attached to the solid support at a 5′-end and comprises a blocking group at a 3′ end that prevents polymerase extension, thereby forming a plurality of tethered amplification products.

15. The method of claim 14 , wherein said solid support comprises a plurality of second oligonucleotides.

16. The method of claim 14 , wherein said solid support comprises an array of wells.

17. The method of claim 14 , wherein said solid support comprises a plurality of splint oligonucleotides randomly distributed on the solid support.

18. The method of claim 14 , wherein said solid support comprises a patterned surface.

19. The method of claim 14 , further comprising sequencing said tethered amplification products on said solid support.

20. The method of claim 19 , wherein sequencing comprises hybridizing one or more sequencing primers to the tethered amplification products and generating one or more sequencing reads by detecting a sequence of signals.

21. The method of claim 1 , wherein the splint oligonucleotide includes a first sequence complementary to a first primer sequence, a second primer sequence, and a third primer sequence, wherein the third primer sequence is between the first and the second primer sequence.

22. The method of claim 21 , wherein the third primer sequence is a sequencing primer binding sequence.

23. A method of amplifying a single-stranded polynucleotide, the method comprising:

contacting a solid support with the single-stranded polynucleotide comprising a first primer binding sequence and a second primer binding sequence, and hybridizing said first primer binding sequence and said second primer binding sequence to a splint oligonucleotide attached to the solid support;

ligating the first primer binding sequence and a second primer binding sequence together to form a circular polynucleotide;

extending the splint oligonucleotide with a polymerase to generate an amplification product comprising a complement of the circular polynucleotide; and

hybridizing the amplification product to a second oligonucleotide comprising a blocking group at a 3′ end that prevents polymerase extension, wherein said second oligonucleotide is attached to the solid support.